Throughout this application, various publications are referenced. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains. Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, and autonomic function and can also induce physical dependence (Koob, et al (1992)). The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal and immune functions (Olson, et al (1989)). Opioids exert their actions by binding to specific membrane-associated receptors located throughout the central and peripheral nervous system (Pert, et al. (1973)). The endogenous ligands of these opioid receptors have been identified as a family of more than 20 opioid peptides that derive from the three precursor proteins proopiomelanocortin, proenkephalin, and prodynorphin (Hughes, et al. (1975); Akil, et al. (1984)). Although the opioid peptides belong to a class of molecules distinct from the opioid alkaloids, they share common structural features including a positive charge juxtaposed with an aromatic ring that is required for interaction with the receptor (Bradbury, et al. (1976)).
Pharmacological studies have suggested that there are at least three major classes of opioid receptors, designated xcex4, xcexa, xcexc and "sgr" (Simon 1991; Lutz, et al. (1992)). The classes differ in their affinity for various opioid ligands and in their cellular distribution. The different classes of opioid receptors are believed to serve different physiological functions (Olson, et al., (1989); Simon (1991); Lutz and Pfister (1992)). However, there is substantial overlap of function as well as of distribution. Despite pharmacological and physiological heterogeneity, at least some types of opioid receptors inhibit adenylate cyclase, increase K+ conductance, and inactivate Ca2+ channels through a pertussis toxin-sensitive mechanism (Puttfarcken, et al. 1988; Attali, et al. 1989; Hsia, et al., 1984). These results and others suggest that opioid receptors belong to the large family of cell surface receptors that signal through G proteins (Di Chiara, et al. (1992); Loh, et al. (1990)).
Several attempts to clone cDNAs encoding opioid receptors have been reported. A cDNA encoding an opioid-binding protein (OBCAM) with xcexc selectivity was isolated (Schofield, et al. (1989)), but the predicted protein lacks transmembrane domains presumed necessary for signal transduction. More recently, the isolation of another cDNA was reported, which was obtained by expression cloning (Xie, et al. (1992)). The deduced protein sequence displays seven putative transmembrane domains and is very similar to the human neuromedin K receptor. However, the affinity of opioid ligands for this receptor expressed in COS cells is two orders of magnitude below the expected value, and no subtype selectivity can be shown.
Many cell surface receptor/transmembrane systems consist of at least three membrane-bound polypeptide components: (a) a cell-surface receptor; (b) an effector, such as an ion channel or the enzyme adenylate cyclase; and (c) a guanine nucleotide-binding regulatory polypeptide or G protein, that is coupled to both the receptor and its effector.
G protein-coupled receptors mediate the actions of extracellular signals as diverse as light, odorants, peptide hormones and neurotransmitters. Such receptors have been identified in organisms as evolutionarily divergent as yeast and man. Nearly all G protein coupled receptors bear sequence similarities with one another, and it is thought that all share a similar topological motif consisting of seven hydrophobic (and potentially a-helical) segments that span the lipid bilayer (Dohlman et al. (1987); Dohlman et al. (1991)).
G proteins consist of three tightly associated subunits, xcex1, xcex2 and xcex3 (1:1:1) in order of decreasing mass. Following agonist binding to the receptor, a conformational change is transmitted to the G protein, which causes the Gxcex1-subunit to exchange a bound GDP for GTP and to dissociate from the xcex2xcex3-subunits. The GTP-bound form of the xcex1-subunit is typically the effector-modulating moiety. Signal amplification results from the ability of a single receptor to activate many G protein molecules, and from the stimulation by Gxcex1-GTP of many catalytic cycles of the effector.
The family of regulatory G proteins comprises a multiplicity of different xcex1-subunits (greater than twenty in humans), which associate with a smaller pool of xcex2- and xcex3-subunits (greater than four each) (Strothman and Simon (1991)). Thus, it is anticipated that differences in the xcex1-subunits probably distinguish the various G protein oligomers, although the targeting or function of the various xcex1-subunits might also depend on the xcex2xcex3 subunits with which they associate (Strothman and Simon (1991).
Improvements in cell culture and in pharmacological methods, and more recently, use of molecular cloning and gene expression techniques have led to the identification and characterization of many seven-transmembrane segment receptors, including new sub-types and sub-sub-types of previously identified receptors. The xcex11 and xcex12-adrenergic receptors once thought to each consist of single receptor species, are now known to each be encoded by at least three distinct genes (Kobilka et al. (1987); Regan et al. (1988); Cotecchia et al. (1988); Lomasney (1990)). In addition to rhodopsin in rod cells, which mediates vision in dim light, three highly similar cone pigments mediating color vision have been cloned (Nathans et al. (1986)A; and Nathans et al. (1986)B). All of the family of G protein-coupled receptors appear to be similar to other members of the family of G protein-coupled receptors (e.g., dopaminergic, muscarinic, serotonergic, tachykinin), and each appears to share the characteristic seven-transmembrane segment topography.
When comparing the seven-transmembrane segment receptors with one another, a discernible pattern of amino acid sequence conservation is observed. Transmembrane domains are often the most similar, whereas the amino and carboxyl terminal regions and the cytoplasmic loop connecting transmembrane segments V and VI can be quite divergent (Dohlman et al. (1987)).
Interaction with cytoplasmic polypeptides, such as kinases and G proteins, was predicted to involve the hydrophobic loops connecting the transmembrane domains of the receptor. The challenge, however, has been to determine which features are preserved among the seven-transmembrane segment receptors because of conservation of function, and which divergent features represent structural adaptations to new functions. A number of strategies have been used to test these ideas, including the use of recombinant DNA and gene expression techniques for the construction of substitution and deletion mutants, as well as of hybrid or chimeric receptors (Dohlman et al. (1991)).
With the growing number of receptor sub-types, G-protein subunits, and effectors, characterization of ligand binding and G protein recognition properties of these receptors is an important area for investigation. It has long been known that multiple receptors can couple to a single G protein and, as in the case of epinephrine binding to xcex22- and xcex12-adrenergic receptors, a single ligand can bind to multiple functionally distinct receptor sub-types. Moreover, G proteins with similar receptor and effector coupling specificities have also been identified. For example, three species of human Gi have been cloned (Itoh et al. (1988)), and alternate mRNA splicing has been shown to result in multiple variants of Gs (Kozasa et al. (1988)). Cloning and over production of the muscarinic and xcex12-adrenergic receptors led to the demonstration that a single receptor sub-type, when expressed at high levels in the cell, will couple to more than one type of G protein.
Opioid receptors are known to be sensitive to reducing agents, and the occurrence of a disulfide bridge has been postulated as essential for ligand binding (Gioannini, et al. (1989)). For rhodopsin, muscarinic, and xcex2-adrenergic receptors, two conserved cysteine residues in each of the two first extracellular loops have been shown critical for stabilizing the functional protein structure and are presumed to do so by forming a disulfide bridge. Structure/function studies of opioid ligands have shown the importance of a protonated amine group for binding to the receptor with high affinity. The binding: site of the receptor might, therefore, possess a critical negatively charged counterpart. Catecholamine receptors display in their sequence a conserved aspartate residue that has been shown necessary for binding the positively charged amine group of their ligands.
Given the complexity and apparent degeneracy of function of various opioid receptors, a question of fundamental importance is how, and under what circumstances do specific sub-type and sub-sub-type receptors exert their physiological effect in the presence of the appropriate stimulatory ligand. A traditional approach to answering this question has been to reconstitute the purified receptor and G protein components in vitro. Unfortunately, purification schemes have been successful for only a very limited number of receptor sub-types and their cognate G-proteins. Alternatively, heterologous expression systems can be of more general usefulness in the characterization of cloned receptors and in elucidating receptor G protein coupling specificity (Marullo et al. (1988); Payette et al. (1990); King et al. (1990)).
One such system was recently developed in yeast cells, in which the genes for a mammalian xcex22-adrenergic receptor and Gs xcex1-subunit were coexpressed (King et al. 1990). Expression of the xcex22-adrenergic receptor to levels several hundred-fold higher than in any human tissue was attained, and ligand binding was shown to be of the appropriate affinity, specificity, and stereoselectivity. Moreover, a xcex22-adrenergic receptor-mediated activation of the pheromone signal transduction pathway was demonstrated by several criteria, including imposition of growth arrest, morphological changes, and induction of ia pheromone-responsive promoter (FUSI) fused to the Escherichia coli lacz gene (encoding xcex2-galactosidase) (King et al. 1990).
Finally, expression of a single receptor in the absence of other related sub-types is often impossible to achieve, even in isolated, non-recombinant mammalian cells. Thus, there has been considerable difficulty in applying the standard approaches of classical genetics or even the powerful techniques of molecular biology to the study of opioid receptors. In particular, means are needed for the identification of the DNA sequences encoding individual opioid receptors. Given such isolated, recombinant sequences, it is possible to address the heretofore intractable problems associated with design and testing of isoform-specific opioid receptor agonists and antagonists. The availability of cDNAs encoding the opioid receptors will permit detailed studies of signal-transduction mechanisms and reveal the anatomical distribution of the mRNAs of these receptors, providing information on their expression pattern in the nervous system. This information should ultimately allow better understanding of the opioid system in analgesia, and also the design of more specific therapeutic drugs.
Availability of polynucleotide sequences encoding opioid receptors, and the polypeptide sequences of the encoded receptors, will significantly increase the capability to design pharmaceutical compositions, such as analgesics, with enhanced specificity of function. In general, the availability of these polypeptide sequences will enable efficient screening of candidate compositions. The principle in operation through the screening process is straightforward: natural agonists and antagonists bind to cell-surface receptors and channels to produce physiological effects; certain other molecules bind to receptors and channels; therefore, certain other molecules may produce physiological effects and act as therapeutic pharmaceutical agents. Thus, the ability of candidate drugs to bind to opioid receptors can function as an extremely effective screening criterion for the selection of pharmaceutical compositions with a desired functional efficacy.
Prior methods for screening candidate drug compositions based on their ability to preferentially bind to cell-surface receptors has been limited to tissue-based techniques. In these techniques, animal tissues rich in the receptor type of interest are extracted and prepared; candidate drugs are then allowed to interact with the prepared tissue and those found to bind to the receptors are selected for further study. However, these tissue-based screening techniques suffer from several significant disadvantages. First, they are expensive because the source of receptor cell tissuexe2x80x94laboratory animalsxe2x80x94is expensive. Second, extensive technical input is required to operate the screens. And, third, the screens may confuse the results because there are no tissues where only one receptor subtype is expressed exclusively. With traditional prior art screens, the wrong interactions are observed or, at best, the proper interactions are observed together with unwanted interactions.
The nucleic acid molecules, proteins and methods of the subject invention overcome the difficulties discussed supra in connection with kappa3 opioid receptors.
The subject invention provides recombinant nucleic acid molecule which encodes a kappa3 opioid receptor.
The subject invention further provides an anti-sense oligonucleotide molecule capable of specifically hybridizing to an mRNA molecule encoding kappa3 opioid receptor, at the portion thereof encoding kappa3 opioid receptor, so as to prevent translation of the MRNA molecule.
The subject invention further provides a nucleic acid molecule encoding the anti-sense oligonucleotide molecule of the subject invention, wherein the nucleic acid molecule is capable of being expressed in a suitable host cell.
The subject invention further provides a vector comprising the recombinant nucleic acid molecule of the subject invention.
The subject invention further provides a host vector system for the production of a kappa3 opioid receptor which comprises the vector of the subject invention in a suitable host cell.
The subject invention further provides a method for producing a kappa3 opioid receptor which comprises growing the host vector system of the subject invention under conditions permitting the production of the kappa3 opioid receptor, and recovering the kappa3 opioid receptor produced thereby.
The subject invention further provides the nucleic acid molecule of the subject invention, wherein the nucleic acid molecule is labeled with a detectable marker.
The subject invention further provides al method for detecting the presence of kappa3 opioid receptor-encoding nucleic acid molecules present in a sample which comprises contacting the sample with the labeled nucleic acid molecule of the subject invention under conditions permitting the labeled nucleic acid molecule to form a complex with kappa3 opioid receptor-encoding nucleic acid molecules present in the sample, and detecting the presence of such complex formed so as to thereby detect the presence of kappa3 opioid receptor-encoding nucleic acid molecules present in the sample.
The subject invention further provides a method for quantitatively determining the amount of kappa3 opioid receptor-encoding nucleic acid molecules present in a sample which comprises contacting the sample with a suitable amount of the labeled nucleic acid molecule of the subject invention under conditions permitting the labeled nucleic acid molecule to form a complex with kappa3 opioid receptor-encoding nucleic acid molecules present in the sample, quantitatively determining the amount of complex so formed so as to thereby quantitatively determine the amount of kappa3 opioid receptor-encoding nucleic acid molecules present in the sample.
The subject invention further provides a purified native kappa3 opioid receptor.
The subject invention further provides the variant kappa3 opioid receptor encoded by the recombinant nucleic acid molecule of the subject invention.
The subject invention further provides a method for obtaining partially purified polyclonal antibodies capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto which method comprises (a) immunizing a subject with the kappa3 opioid receptor encoded by the nucleic acid molecule of the subject invention, (b) recovering from the immunized subject serum comprising antibodies capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto, and (c) partially purifying the antibodies present in the serum, thereby obtaining partially purified polyclonal antibodies capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto.
The subject invention further provides the partially purified antibodies produced by the method of the subject invention.
The subject invention further provides a method for obtaining a purified monoclonal antibody capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto which method comprises (a) immunizing a subject with the kappa3 opioid receptor encoded by the nucleic acid molecule of the subject invention, (b) recovering from the immunized subject a B cell-containing cell sample, (c) contacting the B cell-containing cell sample so recovered with myeloma cells under conditions permitting fusion of the myeloma cells with the B cells therein so as to form hybridoma cells, (d) isolating from the resulting sample a hybridoma cell capable of producing a monoclonal antibody capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto, (e) growing the hybridoma cell so isolated under conditions permitting the production of the monoclonal antibody, and (f) recovering the monoclonal antibody so produced, thereby obtaining a purified monoclonal antibody capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto.
The subject invention further provides the hybridoma cell produced in step (d) of the subject invention.
The subject invention further provides the purified monoclonal antibody produced by the method of the subject invention.
The subject invention further provides an antibody capable of specifically binding to kappa3 opioid receptor and thereby competing with opioid binding thereto, said antibody being labeled with a detectable marker.
The subject invention further provides a method for detecting the presence of kappa3 opioid receptor in a sample which comprises contacting the sample with the antibody of the subject invention under conditions permitting the antibody to form a complex with kappa3 opioid receptor present in the sample, and detecting the presence of complex so formed, so as to thereby detect the presence of kappa3 opioid receptor in the sample.
The subject invention further provides a method for quantitatively determining the amount of kappa3 opioid receptor in a sample which comprises contacting the sample with the antibody of the subject invention under conditions permitting the antibody to form a complex with kappa3 opioid receptor present in the sample, quantitatively determining the amount of complex so formed, and comparing the amount so determined with a known standard, so as to thereby quantitatively determine the amount of kappa3 opioid receptor in the sample.
The subject invention further provides a composition which comprises the antibody of the subject invention in an amount effective to permit imaging cell membrane-bound kappa3 opioid receptor present in a subject, and a pharmaceutically acceptable carrier.
The subject invention further provides a method for imaging cell membrane-bound kappa3 opioid receptor present in a subject which comprises administering to the subject an amount of the composition of the subject invention effective to permit imaging cell membrane-bound kappa3 opioid receptor present in the subject under conditions permitting the antibody of the composition to specifically bind to cell membrane-bound kappa3 opioid receptor present in the subject, and imaging the antibody specifically bound to cell membrane-bound kappa3 opioid receptor present in the subject after a suitable period of time, so as to thereby image cell membrane-bound kappa3 opioid receptor present in the subject.
The subject invention further provides a composition which comprises the antibody of the subject invention in an amount effective to permit quantitatively determining the amount of cell membrane-bound kappa3 opioid receptor present in a subject, and a pharmaceutically acceptable carrier.
The subject invention further provides a method for quantitatively determining the amount of cell membrane-bound kappa3 opioid receptor present in a subject which comprises administering to the subject an amount of the composition of the subject invention effective to permit quantitatively determining the amount of cell membrane-bound kappa3 opioid receptor present in the subject under conditions permitting the antibody of the composition to specifically bind to cell membrane-bound kappa3 opioid receptor in the subject, quantitatively determining the amount of antibody specifically bound to cell membrane-associated kappa3 opioid receptors present in the subject, and comparing the amount so determined with a known standard so as to thereby quantitatively determine the amount of cell membrane-bound kappa3 opioid receptor present in the subject.
The subject invention further provides a method for determining a subject""s potential sensitivity to a kappa3 opioid receptor-specific agent which comprises quantitatively determining the amount of cell membrane-bound kappa3 opioid receptor present in the subject by the method of the subject invention, and comparing the amount of cell membrane-bound kappa3 opioid receptor so determined with the amount of cell membrane-bound kappa3 opioid receptor present in a subject having a known sensitivity to the agent, so as to thereby determine the subject""s potential sensitivity to the kappa3 opioid receptor-specific agent.
The subject invention further provides a method for determining the affinity of an agent for kappa3 opioid receptor which comprises (a) contacting a predetermined amount of kappa3 opioid receptor with (i) a predetermined amount of the agent together with (ii) a predetermined amount of a detectable known ligand of kappa3 opioid receptor, said known ligand being a known ligand of kappa3 opioid receptor with a known affinity therefore, under conditions which would permit the formation of a complex between kappa3 opioid receptor and the detectable known ligand in the absence of the agent, (b) quantitatively determining the amount of kappa3 opioid receptor-detectable known ligand complex so formed, (c) comparing the amount of kappa3 opioid receptor-detectable known ligand complex determined in step (b) with the amount of kappa3 opioid receptor-detectable known ligand complex formed in the absence of the agent, and (d) determining the affinity of the agent for kappa3 opioid receptor based on the comparison in step (c).
The subject invention further provides a, method for determining the affinity of an agent for kappa3 opioid receptor which comprises (a) contacting kappa3 opioid receptor with the agent under conditions which would permit the formation of a complex between kappa3 opioid receptor and a known ligand thereof, (b) contacting a predetermined amount of the agent-receptor complex with a predetermined amount of the antibody of the subject invention under conditions which would permit the formation of a complex between kappa3 opioid receptor and the antibody in the absence of the agent, (c) quantitatively determining the amount of kappa3 opioid receptor-antibody complex so formed, and (d) determining the affinity of the agent for kappa3 opioid receptor based on the amount of kappa3 opioid receptor-antibody complex determined in step (c).
The subject invention further provides a method for determining whether a known ligand of kappa3 opioid receptor is an agonist thereof which comprises (a) contacting the kappa3 opioid receptor with (i) a predetermined amount of the ligand and (ii) a predetermined amount of GTP or analog thereof under conditions which would permit the binding of the ligand to the kappa3 opioid receptor in the absence of GTP or analog thereof, (b) quantitatively determining the percentage of such predetermined amount of ligand bound to the kappa3 opioid receptor, and (c) comparing the percentage so determined with the percentage of such predetermined amount of ligand bound to the kappa3 opioid receptor in the absence of GTP or analog thereof, a lower percentage in the presence of GTP or analog thereof relative to the percentage in the absence of GTP or analog thereof indicating that the ligand is an agonist of kappa3 opioid receptor.
Finally, the subject invention provides a method for determining whether a ligand of kappa3 opioid receptor is an antagonist thereof which comprises (a) contacting the kappa3 opioid receptor with (i) a predetermined amount of the ligand and (ii) a predetermined amount of GTP or analog thereof under conditions which would permit the binding of the ligand to the kappa3 opioid receptor in the absence of GTP or analog thereof, (b) quantitatively determining the percentage of such predetermined amount of ligand bound to the kappa3 opioid receptor, and (c) comparing the percentage so determined with the percentage of such predetermined amount of ligand bound to the kappa3 opioid receptor in the absence of GTP or analog thereof, an equality between the percentage in the presence of GTP or analog thereof and the percentage in the absence of GTP or analog thereof indicating that the ligand is an antagonist of kappa3 opioid receptor.